1. Field of the Invention
This invention relates to a culturing method of living cells and more particularly to a method for a large scale, high concentration culturing, a culturing system and a culturing apparatus of living cells which perform suspension culture of living cells in a liquid medium and efficiently remove waste components.
2. Description of the Prior Art
Recently, the production of a monoclonal antibody and interferon by the culture of animal cells and the production of natural pigments by vegetable cells have been started in addition to the culture of microorganism cells. To produce such useful materials on an industrial basis, so-called "high density culture" by which cells can be cultured on a large scale for a long period of time while keeping them stable under a high concentration becomes necessary.
Nutrient components are consumed while waste components such as lactate and the like are generated in a culture tank during the cell propagation process and if this condition is left standing as such, the propagation of the living cells is impeded and high density culture is not attained. Therefore, so-called "perfusion culture" is essential, according to which separates the cells in the culture broth, removes the cell-free waste solution to the outside of the system and exchanges with a fresh medium.
Since the cell is a kind of particles, which have a greater specific gravity than that of the medium, a perfusion culturing method which applies a heretofore solid-liquid separation technique is known. As the separation methods of cells, sedimentation separation, centrifugal separation and membrane filteration are ordinarily employed. A membrane filteration device is disposed in a culture tank of a culturing apparatus or outside the culturing apparatus. This is described, for example, in Scientific American 248 (1) p. 24 (1983) and in Japanese Patent Laid-Open No. 102187/1985.
The studies of these conventional methods by the present inventors revealed that if a membrane filtration device is disposed in the culturing apparatus, the cells attached to the membrane surface propagate to bring about irreversible clogging. Moreover, once the culture is started, the exchange of the filtration device is not possible during the culturing period and since the structure and position of the filtration device affects greatly mixing the solutions in the culturing apparatus and the supply of oxygen, scale-up of the culture apparatus is difficult to attain.
The latter two problems can be solved by disposing the filtration device outside the culturing apparatus, but it has been found that if the filtration device is simply disposed outside the culturing apparatus and ordinary back-wash is simply carried out, it is difficult to conduct perfusion-culture for a long period of time without clogging of the film particularly under a high concentration of 1.times.10.sup.7 cells/ml or more.